The discovery of substances that control the growth of mammalian cells, especially human cells, and the mechanism by which they work is currently one of the major focuses of biomedical research concerned with tissue repair and wound healing. Fibroblast growth factors (FGFs), mitogens for various cell types including fibroblasts, have been identified and it has been suggested that they may induce mitosis which will result in tissue repair. Fibroblast mitogenic activity was initially observed with extracts of tissue from the central nervous system. Brain-derived fibroblast mitogens were first described by Trowell et al., J. Exp. Biol. 16: 60-70 (1939) and Hoffman, Growth 4: 361-376 (1940). It was subsequently shown that pituitary extracts also had potent mitogenic activity for fibroblasts, Armelin, Proc. Natl. Acad. Sci. USA 70: 2702-2706 (1973). Partial purification of both brain and pituitary fibroblast growth factor revealed copurification of mitogenic activity against a variety of types of differentiated cells including vascular endothelial cells, Gospodarowicz et al., Natl. Cancer Inst. Monogr. 48: 109-130 (1978).
Fibroblast growth factor was originally thought to be a single peptide derived from the limited proteolysis of myelin basic protein. It has recently been shown that FGF exists in two forms, acidic FGF (aFGF) and basic FGF (bFGF), and that both forms can be isolated and purified from mammalian brain, Thomas, et al., Proc. Natl. Acad. Sci. USA 81: 357-361 (1984), Lemmon and Bradshaw, J. Cell Blochem. 21:195-208 (1983). Numerous cell types respond to stimulation with either purified aFGF or bFGF to synthesize DNA and divide, including primary fibroblasts, vascular and corneal endothelial cells, chondrocytes, osteoblasts, myoblasts, smooth muscle cells, glial cells and neuroblasts, Esch et al., Proc. Natl. Acad. Sci. USA 82: 6507-6511 (1985); Kuo et al., Fed. Proc. 44: 695 (1985). Pure bovine brain-derived aFGF not only acts as a potent mitogen for vascular endothelial cells in culture but also induces blood vessel growth in vivo, Thomas, et al., Proc. Natl. Acad. Sci. USA 82: 6409-6413 (1985). The mitogenic activity of purified aFGF can also be used to promote wound healing, Thomas U.S. Pat. No. 4,444,760.
Acidic fibroblast growth factor was originally purified to homogeneity from bovine brain based on its mitogenic activity for BALB/c 3T3 fibroblasts, Thomas et al., Proc. Natl. Acad. Sci. USA 81: 357-361 (1984). This brain-derived growth factor has been repurified and renamed in multiple laboratories based both on its: mitogenic activity for vascular endothelial cells, astroglial cells and prostate epithelial cells (endothelial cell growth factor, astroglial growth factor 1 and prostatropin); source (retinal-derived growth factor, eye-derived growth factor II, brain-derived growth factor); and binding to heparin-Sepharose (class 1 heparin-binding growth factor or heparin-binding growth factor alpha) Thomas and Gimenez-Gallego, TIBS 11:81-84 (1986). The amino acid sequence of bovine aFGF has been determined, recognized to be highly homologous to basic FGF and perhaps related to the fibroblast mitogens interleukin 1-alpha and 1-beta, Gimenez-Gallego et al., Science 230: 1385-1388 (1985). The complete amino acid sequence of human aFGF has been determined from the purified protein, Gimenez-Gallego et al., Blochem. Biophy. Res. Comm. 138: 611-617 (1986), and from the gene, Jaye et al., Science 233: 541-545 (1986). Heretofore the availability of aFGF has been dependent upon the isolation and purification of the proteins from animal tissues, generally bovine. The unavailability of human aFGF has limited the use of aFGF as a therapeutic agent in humans. The present invention will allow the production of therapeutically significant amounts of highly purified human and bovine aFGF.
To date, the growth of vascular endothelial cells could only be accomplished using very high concentrations of fetal calf or adult bovine serum, 10 to 30%. The results were variable, depending on the particular lot of calf serum and the rate of cell growth was generally slow. Now, with brain-derived and recombinant aFGF, rapid endothelial cell growth rates are achieved with serum levels as low as 0 to 2%.
This novel method of reproducible stimulation of vascular endothelial cell growth, mediated by pure brain-derived and recombinant aFGF, permits the covering of synthetic polymeric vessels with non-thrombogenic vascular endothelial cells from a host animal, including human, whereby many or all of the clotting problems associated with synthetic vessel grafts are obviated. Endothelial cell stimulation with aFGF is also used for the production of vessels in vitro by growth of host vascular endothelial cells on tubular supports, for implantation back into the same host animal, including human, whereby immunological rejection of the implant will be obviated and the frequent limited supply of good vessels within the patient for transplant will be obviated. Tubular supports are coated in vitro with aFGF prior to implantation into a host animal. Following implantation endothelial cells migrate into and grow on the artificial surface producing in vivo artificial vessels. Acidic fibroblast growth factor can also be used for the stimulation or facilitation of blood vessel growth and repair in vivo, whereby the flow of blood to tissues deprived of adequate oxygen and/or other blood borne components is increased.